Leveraging a synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells

Abstract

There is an urgent need to develop a more efficacious anti-tuberculosis vaccine as the current live-attenuated vaccine strain BCG fails to prevent pulmonary infection in adults. Our long-term goal is to test whether increasing the immunogenicity of BCG will improve vaccine effectiveness while maintaining its proven safety profile. In this study, we leverage a synthetic biology approach to engineer BCG to produce more (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a phosphoantigen produced as an intermediate of bacterial-but not host-isoprenoid biosynthesis via the methylerythritol phosphate (MEP) pathway. Importantly, HMBPP strongly activates and expands Vγ9Vδ2 T cells, which are unique to higher-order primates and protect against Mycobacterium tuberculosis infection. Prior work to engineer BCG to produce specific ligands and antigens has been attempted to some success; however, our strategy exploits a self-nonself recognition mechanism in the host via HMBPP sensing, which has not been attempted before in this way. To inform the design of our recombinant strains, we performed synteny analyses of >63 mycobacterial species, which revealed that isoprenoid biosynthetic genes are not found in gene clusters or operons across all the 356 surveyed genomes. This analysis also revealed pair biases of isoprenoid biosynthesis genes frequently found in close proximity. In our engineering attempts, we found that simply overexpressing the rate-limiting gene in the pathway was toxic to the bacterium. Thus, we generated synthetic loci with the goal of specifically overproducing HMBPP, and tested the ability of these engineered strains to induce human Vγ9Vδ2 expansion in an in vitro stimulation assay. We found that BCG expressing a rationally-designed, synthetic MEP locus did not enhance Vγ9Vδ2 T cell expansion over the wild-type vaccine strain, suggesting that ectopic expression of multiple MEP genes may result in feedback inhibition of the pathway. However we found that overexpression of the HMBPP synthase GcpE alone potently induced Vγ9Vδ2 T cell expansion and did not result in downregulation of other pathway genes, presenting a successful strategy to accumulate HMBPP and overcome feedback inhibition in this pathway. While much remains to be done to ultimately develop a more efficacious vaccine, our data present a promising system to improve upon the BCG platform. To our knowledge, this is the first work to attempt reengineering of the MEP pathway in BCG to improve vaccine efficacy.

Figure 1:. The MEP pathway is essential in BCG.

A. The MEP pathway of isoprenoid biosynthesis.Shown in green are end products and shunts; shown in red is HMBPP, the activator of Vγ9Vδ2 T cells. Genes encoding enzymatic steps are as follows: dxs, DOXP synthase; dxr, DOXP reductoisomerase; ispD, MEP cytidylyltransferase; ispE, CDP-ME kinase; ispF, MEcPP synthase; gcpE, HMBPP synthase; ispH, HMBPP reductase; idi, isopentenyl diphosphate isomerase. B. Silencing of MEP genes is lethal in BCG. Targeting of dxr and ispH by inducible CRISPR interference was lethal in BCG. A sgRNA targeting the known essential gene rpoB and a non-targeting sgRNA were included as controls. sgRNAs were cloned into the vector pLJR965 carrying CRISPRi machinery and transformed into BCG. Transcriptional repression of target genes was achieved by the addition of ATc. Plates were incubated at 37°C for a minimum of 12 days before counting and imaging. C. Synteny analysis reveals naturally-occurring gene pair biases to leverage in our synthetic platform. Several gene pairs are frequently found in proximity to each other across mycobacteria: dxs1 + ispH; dxr + ispG/gcpE; ispD + ispF; and occasionally ispH + ispG/gcpE. Synteny analysis was performed on 353 mycobacterial genomes using each gene as a query to generate this 7×7 matrix of co-occurrence.

Figure 2:. Generation of a synthetic MEP locus.

A. Designing pCQ88, a synthetic MEP locus. Using gene pairs identified in our synteny analysis, we designed an integrative plasmid carrying an engineered biosynthetic gene cluster comprising all upper-branch MEP genes. Each operon is composed of one pair of genes (green) driven by a strong, unique mycobacterial promoter (blue), preceded by a ribosome binding site (yellow), and followed by either an exogenous or natural terminator (red). B. Genes in the synthetic MEP locus are not strongly overexpressed. No significant differences in individual gene expression exist between WT and BCG+pCQ88. RNA was isolated from WT and pCQ88-expressing BCG and used in a RT-PCR reaction to assess relative expression of each gene in the synthetic locus. Expression data for ispH is included as a control as it was not encoded in the synthetic MEP synthetic locus. N=3 biological replicates in technical triplicate. C, D. The engineered biosynthetic gene cluster pCQ88 does not strongly enhance Vγ9Vδ2 T cell expansion over baseline. There is no significant difference in Vγ9Vδ2 T cell expansion between WT and BCG+pCQ88. Shown is percent expansion of Vγ9Vδ2 T cells by pure HMBPP or lysate from either WT or BCG+pCQ88 at various dilutions (C) and at 1:500 diluted (D) compared to baseline. Data shown include data from three unique PBMC donors.

Figure 3:. Generation of a gcpE overexpression construct.

A. Designing a gcpE OE construct. The pCQ105 construct contains a single-gene expression platform composed of a strong mycobacterial promoter (blue) driving the expression of gcpE (green), which is preceded by a ribosome binding site (yellow) and followed by a terminator (red). B. HMBPP synthase GcpE is strongly overexpressed. Expression of each gene in the MEP pathway was compared between WT and pCQ105-expressing BCG. Compared to WT, gcpE was significantly overexpressed in the engineered strain (Sidak’s multiple comparisons; p<0.0001****). N=3 biological replicates in technical triplicate. C. Overexpression of gcpE significantly enhances Vγ9Vδ2 T cell expansion over WT. Shown is percent expansion of Vγ9Vδ2 T cells by pure HMBPP, 1:500 diluted lysate from either WT or BCG+pCQ105, compared to baseline. There is a significant difference in Vγ9Vδ2 between WT and BCG+pCQ105 (Sidak’s multiple comparisons; p<0.0001**). Shown is one PBMC donor, representative of independent experiments across two unique donors.